Evolution can be viewed as an algorithm wherein a sequence gives rise to variants and a selection performed on that derivative pool allows for the survival of progeny with an incremental enhancement of the selected trait (Daniel C. Dennett, Darwin's Dangerous Idea, Touchstone, New York, N.Y. 1995). Iterative cycles of the process drive the production of increasingly refined embodiments of the selected trait. In popular models of natural evolution the global “fitness” of the organism is the driving selective force. Beginning at the dawn of civilization man has intervened in the process to exert selections on potential food corps and animals, not for the fitness of the organism, but rather for utility to his kind. This is a “directed evolution”.
In recombinant DNA technologies, individual genes can be isolated and expressed in foreign host organisms allowing the controlled production of specific gene products. This ability forms the basis of the biotechnology industry, with applications in medicine, agriculture and various chemical industries (see, e.g., Evens and Witcher, 1993, Ther. Drug Monit. 15(6):514–20; Steve Prentis, Biotechnology: a new industrial revolution, G. Braziller, NY, NY 1984; Symposium on Biotechnology for Fuels and Chemicals, Totowa, N.J.: Humana Press, 1997). With recombinant DNA technologies, and the isolation of individual genes directed evolution procedures can be applied to these isolated genes. The term “directed evolution”, as commonly used, applies to efforts made to improve the characteristics of a gene product with a particular commercial end in mind (Marrs et al., 1999, Curr. Opin. Microbiol 2(3):241–5), although in some instances the term has been applied to groups of genes defining a pathway (Wackett, 1998, Ann. N Y Acad. Sci. 864:142–52).
The first efforts to accomplish this involved the application of various mutagenesis procedures that introduce changes at single, or at times several, residues of the coding sequence (Kuchner and Arnold, 1997, Trends Biotechnol. 12:523–30). Such efforts have reported some success, albeit, limited. The number of potential changes to be explored is immense, vastly exceeding an experimenter's ability to produce and analyze them. It is clear that most changes are detrimental while only rare alterations yield enhancements in desired trait.
More recently, specialized PCR technologies have been applied to the problem of directed evolution (Stemmer, 1994, Proc. Natl. Acad. Sci. 91:10747–51). The most popular version, primerless PCR or so-called sexual PCR, allows for the re-assortment, or “shuffling,” of closely related sequences. Briefly, a set of related gene sequences are fragmented, denatured, allowed to reanneal, and PCR extension is then performed through a number of cycles to reconstruct unit length genes. This process produces novel sequences that are complex permutations of the substrates. This process has proven to produce genes with significantly varied characteristics, and in many instances phenotypes dramatically improved for selected properties (e.g., Chang et al., 1999, Nat. Biotechnol. 8:793–7). In a set of experiments with a related family of β-lactamases, mutagenesis was compared directly with shuffling. The shuffling procedure proven to dramatically enhance resistance to a novel β-lactam (500-fold) where only modest improvements (8-fold) were noted in with mutagenesis alone (Crameri et al., 1998, Nature 391:288–91). Both mutagenesis and re-assortment sample an array of potential variants. When sampling re-assorted variants, the set of sampled sequences contains variants that are composed of sequence stretches that have themselves been “pre-selected,” over evolutionary time scales, for function. This is in contrast to the sequences derived from mutagenesis where the combinations are likely to be encountered for the first time without “pre-selection.” The hypothesized “pre”-selection aspect of this re-assortment procedure may allow for the apparently more productive nature of the so-called shuffling strategy.
Although “gene shuffling” has had some success and can be credited with popularizing the notion that cloned genes can be tailored to provide more useful variants through directed evolution procedures, it has clear limitations that make alternative strategies desirable. For example, one major shortcoming of “shuffling,” or more precisely, random complex permutation sampling, is that information about a particular member of a combinatorial set only becomes accessible when the exact identity of that member is revealed. When complex permutations are sampled randomly, as in so-called gene shuffling, any information about the context of the sample is lost until its identity is revealed, following sequence determination. Furthermore, random permutation sampling through primeness PCR is a process that requires all subsequent iterations to repeat the enzymatic steps of the process: DNA isolation, DNA fragmentation, PCR reconstruction, and product cloning. A faster and more cost-effective procedure would be desirable.
Plasmid-based recombination has previously been used as an approach for producing novel genes (Piotukh et al., 1992, Molekulyarnaya Biologiya 26(4) part 2:601–604) used homologous recombination to construct hybrid metalloproteinases. This approach used direct repeat recombination, a process requiring only a single crossover event. Such recombination can produce novel genetic arrangements, but each round of iteration requires re-cloning of the sequences targeted for the recombination process, and reagents used for one event cannot reused or archived for subsequent procedures. Although a highly efficient process, this type of recombination does not lend itself to combinatorial reassortments or multiuse libraries.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.